Phenazine Cations as Anticancer Theranostics

The biological properties of two water-soluble organic cations based on polypyridyl structures commonly used as ligands for photoactive transition metal complexes designed to interact with biomolecules are investigated. A cytotoxicity screen employing a small panel of cell lines reveals that both cations show cytotoxicity toward cancer cells but show reduced cytotoxicity to noncancerous HEK293 cells with the more extended system being notably more active. Although it is not a singlet oxygen sensitizer, the more active cation also displayed enhanced potency on irradiation with visible light, making it active at nanomolar concentrations. Using the intrinsic luminescence of the cations, their cellular uptake was investigated in more detail, revealing that the active compound is more readily internalized than its less lipophilic analogue. Colocalization studies with established cell probes reveal that the active cation predominantly localizes within lysosomes and that irradiation leads to the disruption of mitochondrial structure and function. Stimulated emission depletion (STED) nanoscopy and transmission electron microscopy (TEM) imaging reveal that treatment results in distinct lysosomal swelling and extensive cellular vacuolization. Further imaging-based studies confirm that treatment with the active cation induces lysosomal membrane permeabilization, which triggers lysosome-dependent cell-death due to both necrosis and caspase-dependent apoptosis. A preliminary toxicity screen in the Galleria melonella animal model was carried out on both cations and revealed no detectable toxicity up to concentrations of 80 mg/kg. Taken together, these studies indicate that this class of synthetically easy-to-access photoactive compounds offers potential as novel therapeutic leads.


S1a. Anion Metathesis
Compounds were converted from hexafluorophosphate salts to chloride salts by stirring over a DOWEX anionexchange resin in distilled water.The DOWEX was then removed by filtration and the filtrate dried in vacuo to yield either 1 2+ or 2 2+ as a chloride salt.

S1b. Singlet Oxygen Quantum Yield Determination
Compounds 1 2+ and 2 2+ were dissolved in acetonitrile as hexafluorophosphate salts and the optical densities adjusted to 0.1 at 355 nm.The samples were then irradiated with a short pulse Nd-YAG laser at 355 nm.Since singlet oxygen fluoresces at 1270 nm, an NIR detector was used at 77 K that measures fluorescence intensity throughout the pulse.This fluorescence intensity was then measured at various pulse powers starting at 20 μJ through to 500 μJ.The measurement is an average of 512 readings taken by the oscilloscope, and an average of five readings was recorded at each power.The amplitudes of the fluorescence at each power were then compared to peerinaphthenone which has a singlet oxygen quantum yield of 100 % in acetonitrile, to give a quantum yield at each power which was then averaged out.

S1c. Determination of Log P
Octanol-water partition coefficients were obtained via the shake-flask method to determine the relative lipophilicity of 1 2+ and 2 2+ .Aqueous stocks of a known concentration were prepared and added to an equal volume of 1-octanol that had been previously saturated with water overnight.After 24 hours with shaking, each phase was recovered and the concentration of compound in each layer was determined by UV-vis absorption spectroscopy.

S1d. X-Ray Crystallography
X-ray quality crystals of the quaternised hexafluorophosphate salt 2 were obtained by vapour diffusion using a concentrated solution in nitromethane with diethyl ether as an antisolvent.
Data for 2(PF6)2 were collected on a Bruker Kappa Apex-II CCD diffractometer utilising a MoKα sealed-tube X-ray source.Reflections were corrected for absorption by empirical methods (SADABS) based upon symmetry equivalent reflections in combination with measurements at varied azimuthal angles. 1,2The crystal structures were determined and refined against F2 values using ShelXT for solution and ShelXL for refinement through the Olex2 program. 3,4Hydrogen atoms were positioned according to calculations with ideal geometries and refined utilising a riding model and isotropic displacement parameters.Non-hydrogen atoms were refined anisotropically.

S2a. Cell Culture
Cell media was purchased sterile from Sigma-Aldrich.A2780 human ovarian cancer cells and A2780CIS human ovarian cisplatin resistant cells were cultured in RPMI-1640 medium.MCF7 human breast cancer cells, T24 human bladder cancer cells and HEK293 human embryonic kidney cells were cultured in DMEM medium.All growth medium was supplemented with 10 % fetal bovine serum (FBS), 2 mM L-Glutamine, 100 IU mL -1 penicillin and 100 μg mL -1 streptomycin and sterile filtered before use.Cell cultures were grown by incubation at 37 ᵒC with 5 % CO2 and were frequently passaged with trypsin when confluency of around 80 % was reached.All cells used were at passage numbers below 50.

S2b. Preparation of Compounds
Stock solutions of compounds were solubilized in 10 % PBS (Phosphate Buffer Saline) and 90 % media.Solutions were then sterile filtered through a 0.22 μm filter prior to use.

S2c. Cytotoxicity (MTT Assay) and Determination of IC50 Values
MTT (3-(4,5-Dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide)) is a yellow tetrazolium salt that is reduced to formazan in metabolically active cells.Therefore, the purple coloured formazan can be dissolved and quantified through absorbance as a measure of cell viability.Cells were seeded in 48 well plates at a density of 5 x 10 4 cells per well and incubated for 24 hours prior to treatment.After 24 hours, compound (10 % PBS: 90 % medium) was added over a concentration gradient from 0.1 to 100 μM with each concentration in triplicate.Some wells were treated with a concentration range of cisplatin as a positive control.Cells were incubated with the relevant concentrations of compound for 48 hours, after which the media was removed and replaced with MTT (0.5 mg mL -1 in serum-free medium) for a period of 30-45 minutes.The MTT solution was then carefully removed and the formazan eluted with isopropanol (130 μL per well).100 μL from each well was transferred into a 96 well plate to enable quantification of cell viability using a plate reader measuring absorbance at 570 nm.An average absorbance was obtained for each concentration and the cell viability was then calculated as a percentage of the untreated negative control wells.A graph of cell viability against concentration was plotted and the IC50 (the concentration at which the cell viability is 50 %) determined by interpolation.The IC50 values are reported as an average of at least 3 independent biological replicates.

S2d. Photocytotoxicity
Cells were grown in a 4 x 48 well plates and seeded at a density of 5 x 10 4 cells per well and incubated for 24 hours.Each plate of cells were then treated with the relevant compounds at a concentration range of 0.1 to 100 μM in triplicate and incubated for a further 24 hours.The compound was then removed and replaced with fresh medium 30 minutes prior to light treatment.One of the four plates remained in the incubator as a measure of dark cytotoxicity.The other three plates were irradiated by a broadband 4000K natural white color light illumination source to provide light doses of 8, 24, 48 J cm -2 respectively.All plates were then incubated for another 24 hours following light treatment.Media was then removed from each well and replaced with MTT (0.5 mg mL -1 in serum-free medium) for 30-45 minutes.The formazan in each well was eluted with isopropanol and transferred to a 96 well plate for quantification by a plate reader as mentioned in section S2c.The cell viability for each concentration was determined by the average absorbance as a percentage of the absorbance of the untreated control wells.The data was then plotted as a graph of cell viability against concentration to determine the IC50 values by interpolation before and after light treatment.

S2e. Mitochondrial Membrane Potential (Δψm) Assay (TMRE Assay)
The mitochondrial membrane potential after treatment with compounds was determined through the TMRE Mitochondrial Membrane Potential Assay Kit purchased from Abcam.This assay relies on TMRE (tetramethylrhodamine, ethyl ester), a cationic dye, accumulating in active mitochondria as a result of their negative charge.Therefore, inactive or damaged mitochondria will exhibit a decreased membrane potential and will no longer accumulate TMRE.A2780 cells were seeded in a 96 well plate at a density of 10 4 cells per well and were incubated for 24 hours prior to treatment.The media was then removed and replaced with the relevant concentration of 1 2+ and 2 2+ and fresh media added to the untreated control wells in triplicate.Cells were then incubated with the compounds for the relevant time points.10 minutes before the addition of TMRE, 20 μM FCCP (carbonyl cyaninde 4-(trifluoromethoxy) phenylhydrazone) was added to additional wells in normal RPMI media.This acts a positive control by causing depolarization or mitochondrial membranes, meaning that TMRE is no longer accumulated.Cells were then incubated with 500 nM TMRE for 20 minutes.After the incubation time was complete, cells were washed with PBS and then fresh PBS added to each well and the fluorescence recorded on a plate reader using excitation and emission of 544nm and 590 nm respectively.

S2f. Single Stain Live Cell Microscopy
Live cell images were prepared by seeding cells at a density of 6 x 10 4 onto a 35 mm imaging μ-dish with an ibidi polymer coverslip bottom and allowed to adhere with 24 hours incubation.The media was then removed, the cells washed once with PBS and the corresponding concentration of compound added for the relevant time points.Prior to imaging, media was removed and the cells washed once with PBS and twice with media before replacing with fresh media.Samples were then imaged immediately.Imaging was performed using an Airyscan confocal laser scanning inverted microscope ZEISS LSM 880 with an environmental control chamber.Images were taken with an oil immersion 63 x objective using 518 F immersion media.The 405 nm excitation laser was used to excite the compound and luminescent images were collected between 500-600nm.Detector gain and laser power were adjusted to avoid saturation.Images were processed and analysed with FIJI Image J software.

S2g. Co-stain Microscopy with LysoTracker® Deep Red
Cells were grown as previously stated in live cell imaging μ-dishes.After 24 hours media was removed and replaced with 500 nM LysoTracker Deep Red (Life Technologies) in complete media and incubated for 30 minutes.The cells were then washed three times with media and replaced with the relevant concentration of compound (IC50 or below) and then incubated for the corresponding time point (24 hours) at 37 ᵒC and 5 % CO2. 30 minutes prior to imaging the compound was removed and washed with PBS followed by two washes with media.The cells were then imaged immediately in an environmental control chamber by confocal laser scanning microscopy using a ZEISS Airyscan LSM 880.Images were taken using the oil immersion 63 x objective.The compound was excited at 405 nm and emission collected between 500-600 nm and the LysoTracker Deep Red was excited using the 633 nm laser and the emission collected above 650 nm.The images were processed and the colocalisation analysed using FIJI Image J software.

S2h. Co-stain Microscopy with MitoTracker® Deep Red
Cells were grown as previously stated in live cell imaging μ-dishes.After 24 hours media was removed and replaced with the relevant concentration of compound (IC50 or below) and then incubated for the corresponding time point (24 hours) at 37 ᵒC and 5 % CO2.After the incubation period, the media was removed and the cells washed with PBS followed by two washes with media 30 minutes prior to imaging. 1 μM MitoTracker Deep Red (Invitrogen) was then added to the cells and incubated for a further 30 minutes.The cells were then washed three times with media and the samples imaged immediately by confocal laser scanning microscopy using a ZEISS Airyscan LSM 880 in an environmental control chamber.Images were taken using the oil immersion 63 x objective.The compound was excited at 405 nm and emission collected between 500-600 nm and the MitoTracker Deep Red was excited using the 633 nm laser and the emission collected above 650 nm.The images were processed and the colocalisation analysed using FIJI Image J software.

S2i. Co-stain Microscopy with DRAQ5 TM
Cells were grown as previously stated in live cell imaging μ-dishes.After 24 hours media was removed and replaced with the relevant concentration of compound (IC50 or below) and then incubated for the corresponding time point (24 hours) at 37 ᵒC and 5 % CO2.After the incubation period, the media was removed and the cells washed with PBS followed by two washes with media 30 minutes prior to imaging. 1 μM DRAQ5 (Thermo Scientific) was then added to the cells and incubated for a further 10 minutes.The cells were then washed three times with media and the samples imaged immediately by confocal laser scanning microscopy using a ZEISS Airyscan LSM 880 in an environmental control chamber.Images were taken using the oil immersion 63 x objective.The compound was excited at 405 nm and emission collected between 500-600 nm and the DRAQ5 TM was excited using the 633 nm laser and the emission collected above 650 nm.The images were processed and the colocalisation analysed using FIJI Image J software.

S2j. Co-stain Microscopy with ER-Tracker TM Red (glibenclamide BODIPY® TR)
Cells were grown as previously stated in live cell imaging μ-dishes.After 24 hours media was removed and replaced with the relevant concentration of compound (IC50 or below) and then incubated for the corresponding time point (24 hours) at 37 ᵒC and 5 % CO2.After the incubation period, the media was removed and the cells washed with PBS followed by two washes with media 30 minutes prior to imaging. 1 μM ER-Tracker Red (Invitrogen Molecular Probes) was then added to the cells and incubated for a further 20 minutes.The cells were then washed three times with media and the samples imaged immediately by confocal laser scanning microscopy using a ZEISS Airyscan LSM 880 in an environmental control chamber.Images were taken using the oil immersion 63 x objective.The compound was excited at 405 nm and emission collected between 500-580 nm and the ER-Tracker Red was excited using the 561 nm laser and the emission collected between 600-650 nm.The images were processed and the colocalisation analysed using FIJI Image J software.

S2k. Single Stain Fixed Cell Microscopy
Glass cover slips (22 mm x 22 mm) were sterilised and placed into six well plates.A2780 cells were then seeded onto coverslips with RPMI media and incubated for 24 hours at 37 ᵒC, 5 % CO2.After 24 hours, cells were treated with the relevant concentration of [1] and incubated further for the corresponding time point.Cells were then washed twice with RPMI media followed by two washes with PBS and fixed with 4 % PFA (paraformaldehyde) for 20 minutes.The PFA was removed and any remaining PFA was quenched by washing with ammonium chloride solution (50 mM).Before mounting, cells were washed once more with PBS and then coverslips were mounted onto glass slides using mounting medium (ProLong Gold Antifade).Coverslips were sealed using nail varnish and imaged on a LEICA SP8 3X gSTED SMD confocal microscope.

S2l. Transmission Electron Microscopy (TEM)
A2780 cells were seeded in 60 mm dishes and allowed to adhere for 24 hours, after which the media was removed and the relevant concentration of each compound added and the cells were for the corresponding time points.The media was removed and the cells washed with PBS and trypsinised by incubation with trypsin for two minutes.Media was added to inactivate the trypsin and cell suspensions transferred to 10 mL sterile universal tubes for centrifugation to pellet the cells.The media was removed from the pellets and the cells were concentrated by resuspension into 1 mL of media and transferred to sterile Eppendorf tubes.The cell samples were then re-pelleted and fixed with 3 % glutaraldehyde.Osmium tetroxide (OsO4) was applied as a 2 % aqueous solution for 2 hours, after which the samples were dehydrated with several ethanol (70-100 %) washes.

S2m. Cytometry Methods
To investigate dqPPN-induced cell death in the absence of light, cells were grown in 6-well plates and seeded at a density of 1,000,000 cells per well and incubated for 24 hours.Cells were then treated with 2 2+ at 1 μM or 20 μM, and incubated for either 2h (20 μM), 5h (20 μM) or 24h (1 μM and 20 μM).To observe cell death in the presence of light, cells were seeded at a density of 500,000 cells per well in a 6-well plate and incubated for 24h, prior to adding 1 μM 2 2+ for another 24h.The compound was then removed and replaced with fresh medium 30 minutes prior to light treatment.The plate was then irradiated for 30 minutes at a light dose of 48 J cm -2 .Cells were then incubated for 1h post-light.As a positive control for oncosis, cells were seeded 500,000 in a 6-well plate and incubated for 48h.Cells were then washed with PBS, trypsinized, resuspended in fresh media, and subjected to 60℃ for 45min in a water bath before harvesting for analyses.
To determine DNA content of cells post-treatment, cells were washed with PBS, trypsinized and resuspended in DMEM.Cells were then centrifuged at 150xg for 3 mins, the supernatant removed and cells washed in anticlumping buffer (PBS + 2mM EDTA) twice.Cells were then fixed with 70% ethanol overnight at 4℃.DNA was then stained using 5 μM DRAQ5 in anti-clumping buffer overnight at 4℃.Cells were analyzed using an Attune TM NxT flow cytometer (ThermoFisher) using YL4 channel.
To determine whether cells were apoptotic, light-treated cells were washed with PBS, trypsinized and resuspended in DMEM.Cells were then centrifuged for 3 min at 150xg and the supernatant removed.Cells were resuspended in PBS containing 200 nM ApoTracker TM Green (BioLegend, #427402) and 10 μM DRAQ5 and incubated at room temperature for 15m.Cells were then washed twice in anti-clumping buffer before analysis using an Attune TM NxT flow cytometer using YL4 channel (DRAQ5) and BL1 channel (ApoTracker).Dark treated cells were only analyzed using DRAQ5 and size scattering due to the levels of luminescence from the compound at higher concentrations.

S2n. Galleria Mellonella (Wax Moth Larvae) Toxicity Screen
TruLarv Galleria Mellonella were purchased and used for this study and all larvae were a similar weight.For each concentration of compound, 6 Galleria were used and 6 were used for each control condition.The larvae were injected on the first day into their left pro-leg with 10 μL of the appropriate concentration of either [1] or [2] or water employed as an injection control.After injection, the larvae were kept at room temperature in a petri dish lined with filter paper.At each time point (0, 24, 48, 72, 96 and 120 hours) the health each larvae was analysed through three different tests.Melanization was measured on a scale ranging 1-4 where 0 = completely black, 1 = black spots, 2 = tail/line black and 4 = no signs of melanisation.Activity scores were also measured on a numerical scale ranging from 1-3, where 0 = no movement, 1 = larvae corrects itself, 2 = movement with stimulation and 3 = movement without stimulation.The numbers of larvae live or dead were recorded and plotted as a Kaplain-meier survival curves.Following the screen, larvae were disposed of in a humane manner.S1.Log P values for 1 2+ and 2 2+ obtained via the shake flask method determined after 24 hours at room temperature.S3n.Galleria Mellonella Toxicity Screen at 37 ᵒC.

Figure S2 .Figure S5 .Figure S6 .
Figure S2.Cell viability data for HEK293 embryonic kidney cells treated with compounds 1 2+ and 2 2+ 48 hours.Cisplatin was employed for comparison and the experiment performed in triplicate and data given as an average of 3 independent experiments.

Figure S7 .
Figure S7.Live imaging of A2780 cells over a 30 second time course after treatment with 25 μM 2 2+ showing cell swelling and membranes bursting from left to right.(Red square shows membrane splitting).

Figure
Figure S9.A) Effect of 1 2+ and 2 2+ on mitochondrial membrane potential analyzed by TMRE assay.A2780 cells were exposed to IC50 concentrations of either compound for different time points.Prior to TMRE analysis, cells were treated with 20 µM of FCCP for 20 minutes as a positive control.Treatments were performed in triplicate.

Figure S10 .
Figure S10.Effect of 2 2+ with light treatment on mitochondrial membrane potential analysed by TMRE assay.A2780 cells were exposed to different concentrations of 2 2+ for 24 hours followed by light irradiation.Prior to the TMRE analysis, cells were treated with 20 µM of FCCP for 20 minutes as a positive control.

Figure S13 .
Figure S13.Electron microscopy of A2780 cells after treatment 5 μM 2 2+ followed by light irradiation showing A) a dead cell with complete loss of morphology and no structures resembling mitochondria and B) vacuolization of the cytoplasm.

Figure S15 .
Figure S15.Controls for galectin puncta formation immunofluorescence assay in PFA fixed cells stained with an Anti-LGALS3 antibody.Left: untreated A2780 cells (LMP negative control) and right: cells treated with 2 mM LLOMe for 2 h (LMP positive control).

Figure S17 .
Figure S17.Galleria melonella toxicity screen at 37 ᵒC showing Kaplan-Meier survival curves (top) or larvae treated with 0-80 mg/kg of 2 2+ and incubated at 37 ᵒC for 120 hours.Activity score data from the larvae collected every 24 hours and melanisation score data collected every 24 hours (bottom).